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Abstract

The separation of N-acetyl- α -D-galactosaminidase (Azyme.)
from known contaminants (β -galactosidase and β-N-acetylglucosaminidase)
is of interest because it enzymatically cleaves
the terminal N-acetylgalactosamine from type A red blood cells,
giving them type O(H) properties. Analytical isoelectric .
focusing in polyacrylamide and Sephadex gels was employed as
a means of purifying the Azyme. In gels containing wide range
carrier ampholytes, the Azyme.ahd contaminants co-focused but
not at the Azyme isoelectric point. Modifications of focusing
conditions were undertaken in order to change the behavior of
Azyme such that it would separate from the contaminants. The
apparent isoelectric point (apI) of Azyme shifted between pH
4.0 and pH 5.0 with alternations in focusing conditions. Variations in gel and carrier ampholyte types and concentrations
caused only minor Azyme apI shifts. Addition of different
amounts of product inhibitor to the gel caused a slight acidic
shift. In each case examined the apI was independent of the
duration of focusing. Refocusing Azyme did not alter the apI.
The Azyme remained contaminated to some degree in all cases.
The anomalous behavior of Azyme under different conditions
suggested that the Azyme became insoluble before it reached
its pl. Addition of 0.25 KC1 to the electrolytes kept the
Azyme solubilized throughout the focusing procedure, allowing
it to migrate to its pl. At its pl. however, the Azyme was
irreversibly inactivated.